EP1660568B1 - Hydrophobic object comprising a grid of hydrophilic regions, production of said object, and use of the same - Google Patents

Abstract

The invention relates to a plastic object having a plane surface provided with a plurality of hydrophilic regions that are separated from each other by hydrophobic barriers. The invention also relates to methods for producing said object and to uses of the same.

Description

Collections of large numbers of different probes or probes or test compounds ordered / bound / immobilized on a flat surface are referred to as arrays in the scientific terminology. Such arrays allow rapid simultaneous assay / assay of all probes / probe molecules / test compounds by interaction analysis with one or a mixture of analytes in biological samples, ie target biomolecules. The advantage of an array compared to the simultaneous test / assay with immobilized probes or probe compounds on moving elements, such as beads, is that in an array the type (chemical structure and / or identity) of the immobilized Probes or probe molecules or test molecules exactly by the location in the array surface is known and a local test signal (this can be caused by interaction with the target biomolecule, eg by binding, or eg by enzymatic conversion of the probe through the target biomolecule disappear and thus serve directly for the detection) can thus be assigned immediately to a type of molecule. In particular in miniaturized form, arrays of biological probes or probe molecules or test molecules are also called biochips.

• Nukleinsäure Arrays aus DNA-Fragmenten, cDNAs, RNAs, PCR-Produkten, Plasmiden, Bakteriophagen, synthetischen PNA-Oligomeren, welche mittels Hybridisierung (Bildung eines Doppelstrangmoleküls) zu komplementären Nukleinsäureanalyten ausgelesen werden;
• Protein-Arrays aus Antikörpern, in Zellen expremierten Proteinen, Phagen-Fusionsproteinen ("Phage Display") und
• Verbindungsarrays aus synthetischen Peptiden, deren Analoga wie Peptoide, Oligo-Carbamate usw. oder allgemein organisch chemischen Verbindungen, welche beispielsweise mittels Bindung zu affinen Protein- oder anderen Analyten oder beispielsweise mittels enzymatischer Umsetzung ausgelesen werden.

Important examples of such arrays are:

• Nucleic acid arrays of DNA fragments, cDNAs, RNAs, PCR products, plasmids, bacteriophages, synthetic PNA oligomers, which are read by hybridization (formation of a double-stranded molecule) to complementary nucleic acid analyte;
• Protein arrays of antibodies, expressed in cells proteins, phage fusion proteins ("phage display") and
• Compound arrays of synthetic peptides, their analogs such as peptoids, oligo-carbamates, etc., or generally organic chemical compounds which are read, for example, by binding to affine protein or other analytes or, for example, by enzymatic conversion.

• durch einmaliges Verteilen der Lösungen vorgefertigter Sonden bzw. Sondenmolekülen bzw. Testverbindungen auf der Oberfläche
• durch wiederholte serielle Verteilung der Lösungen von Bausteinen für die chemische Synthese der Sonden bzw. Sondenmolekülen bzw. Testverbindungen in situ auf der Oberfläche.

Such arrays are currently produced according to two different principles by placing the probes or probe molecules or test compounds on already prepared material surfaces (an overview gives S. Wolfl in: transcript Laborwelt 2000, 3, 13-20 ):

• by once distributing the solutions of prefabricated probes or probe molecules or test compounds on the surface
• by repeated serial distribution of the solutions of building blocks for the chemical synthesis of the probes or probe molecules or test compounds in situ on the surface.

The surface with the bound probe molecules is then brought into contact with the solution of the target biomolecules over the entire surface, whereby, for example, an optical or radioactive signal is generated when specific interaction has occurred, which is then read out. Mostly such arrays are used only once. If the attachment of probe molecules to the surface were covalent and stable, then, after washing away the bound target biomolecules with detergents or other more specific reagents / competitors, such an array could be reused. This is not possible in today's, almost exclusively based on glass arrays, because the Si-O-R bonds used are sensitive to hydrolysis.

• Trennung von polyklonalen Antiseren in monospezifische epitopgerichtete Subpopulationen ( Valle M, Kremer L, Martinez C, Roncal F, Valpuesta JM, Albar JP, Carrascosa JL (1999) Domain architecture of the bacteriophage Φ29 connector protein, J Mol Biol 288:899-909 )
• Trennung von Protein- oder Nucleinsäuregemischen in ligandspezifische Subpopulationen
• Trennung von proteinpräsentierenden Bakteriophagen in ligandspezifiasche Subpopulationen ( Epitope-targeted proteome analysis: Towards a large-scale automated protein-proteininteraction mapping utilizing synthetic peptide arrays - Bialek, K., Swistowski, A. and Frank, R. (2003) Analytical and Bioanalytical Chemistry, Band 376: 1006.-1013 ).

In addition, this type of use of the arrays is limited only to the detection of the presence and differential quantification of the target biomolecules in the analyte mixture (biological sample). However, many applications would benefit greatly from an array format if the target molecules could be eluted locally and further used or analyzed. Such applications may be referred to as multiple affinity enrichment experiments. Examples are:

• Separation of polyclonal antisera into monospecific epitope-directed subpopulations ( Valle M, Kremer L, Martinez C, Roncal F, Valpuesta JM, Albar JP, Carrascosa JL (1999) Domain architecture of the bacteriophage Φ29 connector protein, J Mol Biol 288: 899-909 )
• Separation of protein or nucleic acid mixtures into ligand-specific subpopulations
• Separation of protein-presenting bacteriophages into ligand-specific subpopulations ( Epitope-targeted proteome analysis: Towards a large-scale automated protein-protein interaction mapping using synthetic peptide arrays - Bialek, K., Swistowski, A. and Frank, R. (2003) Analytical and Bioanalytical Chemistry, Vol. 376: 1006-1013 ).

So far, such a multiple affinity enrichment is only possible if the array before, but at the latest after the incubation with the biological sample is decomposed into site-selective parts and from these the target biomolecules are separately eluted separately. This division is necessary because otherwise the added elution reagent would pass or diffuse from one location to the neighboring locations and thus intermix the detached target biomolecules.

For subsequent isolation of the target biomolecules from the array, while retaining the information to which probe molecule has been preferentially bound, it is e.g. In an array on cellulose, it is necessary to cut this array to isolate the target biomolecules from each probe molecule individually. However, this array is completely destroyed and no longer useful for further simultaneous analysis of complex biochemicals. A corresponding division of arrays on glass substrates would be much more complicated and technically hardly feasible. Automating the process of decomposition and separate elution would also be very costly because it involves complicated mechanical steps.

The object of the invention is a structured functionalized plastic surface for the automated parallel in situ synthesis or immobilization of a variety 5 of probe molecules, which allows their subsequent application for the automated simultaneous affinity enrichment and isolation of biological analyte molecules from complex mixtures.

Thus, the problem underlying the invention is achieved by a

Object from a hydrophobic plastic, and having a planar surface which is provided with a plurality of hydrophilic areas separated from each other by hydrophobic barrier / webs of the unmodified plastic surface whose extension is ¹ / ^_10-10 / ₁₀ of the smallest dimension of a hydrophilic region, are delimited, wherein the plastic surface has been subjected to the immobilization or binding of probe molecules for functionalization with hydroxy groups of a plasma treatment or ozonolysis.

In the subject invention, the plurality of hydrophilic regions and the hydrophobic barriers can form a grid.

The article of the invention may be polyethylene (PE), polypropylene (PP), polymethylpentene (PMP), polytetrafluoroethylene (PTFE, Teflon) or polyamide.

The article of the invention may be colored.

In the case of the article according to the invention, the plastic may have a content of inorganic material, in particular graphite, and / or organic material, in particular of inert inorganic and / or organic material.

The article according to the invention can be provided as a flat material, which is optionally applied to a carrier.

The article according to the invention may be provided as a film.

In the subject matter of the invention, the size of each hydrophilic region may correspond to a liquid volume dispensable by a pipette and wetting the region or the size of a living cell.

In the subject of the invention, the hydrophilic regions may carry probe molecules suitable for simultaneous affinity enrichment and isolation of analyte molecules.

In the subject of the invention, the probe molecules may be immobilized on the hydrophilic regions or bound to the hydrophilic regions.

In the case of the article according to the invention, the plastic surface can be functionalized for the immobilization or binding of probe molecules, in particular be provided or provided with functional groups.

For functionalization, a mask may have been provided which corresponds to the hydrophobic barriers to be provided or predetermines these hydrophobic barriers, and the masked object, insofar as it has not been masked, may have a mask Have been subjected to functionalization to form the hydrophilic regions.

In the article according to the invention, the plastic surface with hydroxy, amino (especially NH ₂ groups), carboxyl, aldehyde or keto groups may be provided or provided as functional groups.

In the case of the article according to the invention, the surface may be provided with 0.5 to 15 and in particular 0.1 to 13.5 nanomole hydroxy groups / cm ² .

In the article according to the invention, the plastic surface may be further provided with functional groups by grafting a functionalized polymer layer onto the functionalized plastic surface.

The grafting may have been carried out in the absence of crosslinkers, in particular of bifunctional monomers.

In the subject matter of the invention, the probe molecules may have been applied to the functionalized plastic surface by synthesis, by immobilization or by adhesion.

In the subject of the invention, the probe molecules may have been applied by a step synthesis.

In the case of the article according to the invention, the probe molecules can be immobilized by chemical bonding or have been immobilized, in particular via a linker.

In the case of the article according to the invention, the probe molecules can not be applied to be washable adhesively or have been applied.

In the subject matter according to the invention, peptides, peptide analogues, in particular peptoids and / or peptomers, PNA, oligonucleotides, DNA, organic molecules, natural substances and / or oligosaccharides can be or have been applied as probe molecules.

In the subject matter of the invention, the probe molecules may be different from the hydrophilic region to the hydrophilic region, or different probe molecule mixtures may be provided from the hydrophilic region to the hydrophilic region.

In the case of the article according to the invention, a planar surface may have a size in the range from 1 mm ² to 1 m ² , in particular from a few mm ² to a few cm ² .

Furthermore, the object underlying the invention is achieved by a process for the production of a plastic article having a planar surface provided with a multiplicity of hydrophilic regions which are delimited from one another by hydrophobic barriers and which has been functionalized for feeding with probe molecules, starting from a hydrophobic plastic, providing the object with a mask which corresponds to the hydrophobic barriers to be provided or pretending these hydrophobic barriers, and functionalizing the object, if it has not been masked, by hydrophilizing it.

In the method according to the invention can be hydrophilized by providing hydroxy, amino, carboxyl, aldehyde or keto groups.

In the process according to the invention, it is possible to subject the partially hydrophilized article to a graft polymerization, which in turn provides hydroxy, amino, carboxyl, aldehyde or keto groups.

In the inventive method for producing an article around a hydrophobic plastic you can provide the object with probe molecules.

Finally, the problem underlying the invention is solved by using an article according to the invention for a simultaneous affinity enrichment or affinity purification and isolation of analyte particles, in particular of biological analyte particles, preferably Molecules, proteins, antibodies, nucleic acids, phages, oligosaccharides or cells.

In the use according to the invention, after the affinity enrichment or affinity purification, an elution solution can be applied individually per hydrophilic area and the respective analyte particle isolated.

In the use according to the invention, the affinity enrichment or affinity purification and / or the isolation of the analyte particles can be carried out automatically.

In the use according to the invention, the object freed from analyte particles can be used again for affinity enrichment or affinity purification.

According to the invention, therefore, homogeneously hydrophilic regions / patches are applied to a homogeneously hydrophobic plastic surface, which are then sufficiently separated from one another by the hydrophobic barriers / webs of the unmodified plastic surface. The size ratios of the hydrophilic and hydrophobic regions are selected such that a defined volume of liquid can be pipetted onto a hydrophilic region at any time and this volume automatically and completely fills this hydrophilic region with the form which can be freely selected by the process described here, and not flows over the hydrophobic areas. The liquid volumes that can be used for each hydrophilic area are given by the complex ratios of the respective surface tensions. These volumes can be but can be easily determined experimentally by first covering the entire structured plastic surface with an excess of liquid and then allowing the excess to flow off by slight inclination; on the hydrophilic areas, the volumes remain adhered by wetting and can be measured after careful removal. This very simple "auto-filling" of the hydrophilic regions can also be used advantageously in many steps of the affinity enrichment procedures. On these hydrophilic patches, the probe molecules are later synthesized or immobilized in situ. An overview of the micro- and nanostructuring of surfaces for the production of hydrophobic and hydrophilic structures can be found in the article " Softlithography "by Xia, Y., Whitesides, GM, Angewandte Chemie, 110, 568 (1998 ). Due to the hydrophobic / hydrophilic structured areas / patches on the plastic surface, after an affinity enrichment of the target biomolecules, isolation of these target biomolecules from each individual patch is possible individually without destroying the array because the volume of elution solution dispensed does not vary from one area. Patch can flow to the neighboring. The array is thus reusable. Since the individual probe molecules are delimited from one another by the hydrophobic barriers / bridges, the elution of the target biomolecules can easily be automated. The elution solution can be applied with a pipette / pipetting robot and resumed after a corresponding exposure time with the same pipette / pipetting robot. This is spatially resolved for each individual probe molecule.

Known methods make use of such hydrophobic / hydrophilic structures in order, for example, to be able to place a large number of different molecules specifically on predetermined points of a surface and to be able to analyze these molecules later by means of mass spectrometry in the form of MALDI ( GB 2 332 273 ). Here are used as a basis metals such as silicon wafers or gold-coated targets. The hydrophilic areas (dots) are produced by applying small drops of hydrophilic lacquers. The probe molecules are deposited on these structures by binding to nanoparticles, which then collect at the hydrophilic areas of the surface. Such surfaces are not intended and suitable for solid covalent synthesis or immobilization.

Hydrophilic surfaces on which hydrophobic coatings are coated with hydrophobic coatings are available in WO 98/03257 described for the parallel implementation of miniaturized test reactions in small, parallel liquid droplets. Photolithographic methods for applying structured areas to glass substrates using positive or negative photoresists are also known in US Pat WO 94/27719 described.

Also, known arrays based on three-dimensional polyacrylamide gel patches have always been produced on a glass surface. See also: " Manual Manufacturing of Oligonucleotides, DNA, and Protein Microchips "- Guschin, D. et al., (1997), Analytical Biochemistry, 250, 203-211 , Such 3D gel patch arrays are produced by solution polymerization of suitable acrylamide monomers between two glass plates and chemically bonded on one side. However, these glass surfaces exhibit the above-mentioned sensitivity to hydrolysis and are therefore useful only once for testing. In addition, these polyacrylamide gel arrays have not been used for the chemical in situ synthesis of the probe molecules. The plastic surfaces / foils developed by us are stable against many aggressive chemical reagents and at an aqueous pH of 0 to 14. Immobilized or bound to grafted on patches probe molecules can not be detached with aqueous washing / regeneration solutions of the plastic surface / film. Therefore, our plastic surface / film based arrays can be used multiple times.

The invention will be explained in more detail by examples and an illustration. illustration 1 shows: A polypropylene film with a grid of rectangular graft polymerized patches (2 x 2 mm). The patches of the right half and the highlighted area are wetted with water / glycerol (1: 1 / V: V).

Modification of plastic surface / foil

For the synthesis or covalent immobilization of probe molecules, functional groups must be generated on the plastic surface.

One way of functionalizing plastic surfaces is the direct functionalization of polymer surfaces with the aim of generating hydroxy or amino groups and has already been described in detail in the patent "Apparatus for effectively generating freely definable repertoires / libraries with large numbers of ligand and acceptor structures for combinatorial synthesis and to ultrafast test performance in biological, biochemical, pharmaceutical, medical and chemical drug discovery "- Frank, R., Zander, N., Melberg, Y., Gausepohl, H., Schneider-Mergener, J. Adler, F., Türk, G (Applicant: ABIMED Analysiertechnik GmbH, GBF, Jerini Biotools GmbH, MediUm Tech GmbH) P (1) 98 18 999.0, PCT / EP 99/02 811 and described in the literature cited therein.

1. 1. Oxidation von PP, PE, PMP mit Chromtrioxid, Ozon, Wasserstoffperoxid/Trifluoressigsäure
2. 2. Eliminierung von Fluorid aus PVDF
3. 3. Reduktion von PTFE, PCTFE mit Naphtalin/Natrium oder Benzoin
4. 4. Verseifung von EVA
5. 5. Zusätzliche Hydroborierung von unter 1. - 3. beschriebenen Materialien
6. 6. Direkte Hydroxylierung oder Aminierung von PP, PE, PMP mit Radiofrequenzplasma

In it, the following procedures were listed, which are also applicable to the above-mentioned plastic surfaces:

1. 1. Oxidation of PP, PE, PMP with chromium trioxide, ozone, hydrogen peroxide / trifluoroacetic acid
2. 2. Elimination of fluoride from PVDF
3. 3. Reduction of PTFE, PCTFE with naphthalene / sodium or benzoin
4. 4. Saponification of EVA
5. 5. Additional hydroboration of materials described under 1. - 3.
6. 6. Direct hydroxylation or amination of PP, PE, PMP with radio frequency plasma

Some of these methods have also been used for structuring modification of plastic surfaces ( WO 99/58245 ). Particularly suitable for the chemical synthesis and immobilization of probe molecules are surface modifications with primary amines, because there are a variety of reagents with which probe molecules or synthetic building blocks can be covalently linked to such amino functions. Reactions for the introduction of amino groups by means of carboxyl or hydroxyl functions are known and are used here ("Method for producing polymeric solid phase supporting materials" - Ulbricht, M., Volkmer-Engert, R., Germeroth, L., Wenschuh, H., (Applicant: Jerini Biotools GmbH) PCT application WO 00/12575 ; "Derivatized Polymeric Materials - Frank, R., Matysiak, S. (Applicant: GBF) P (1) 96 38 085.5, PCT / EP97 / 05123 ; US 269,002 ; EP 97 943 867.8 ; Korea 10-1999-7002279 ; Norway 99 1296 ; Australia 97.45 553 ).

Another possibility for the functionalization of plastic surfaces is the graft polymerization. It is on the plastic surface / film (base polymer) another functionalized. Applied polymer layer. The following described methods have been used successfully for this: For a review of the photoinitiators used in W-induced graft polymerization, see Fouassier, J.P., Photoinitiation, Photopolymerization and Photocuring, Hansers Publishers, Munich, Vienna, New York, 1995. For a review of the monomers used to functionalize polymers by photopolymerization, see WO 00/12575 , These monomers are suitable for all the graft polymerization processes listed below.

A process for the graft polymerization of, for example, acrylic acid monomers onto polyethylene moldings by gamma-ray initiation was carried out in WO 84/03564 described and used for the production of plastic carriers for the synthesis and testing of peptides. A corresponding process for polypropylene molded articles has been disclosed in P (1) 98 18 999.0. However, all of these methods have hitherto been used only for a modification which does not structure in our sense. A special case describes WO 02/066984 A2 , Smallest bumps (roughness, pixels, smaller than 1/10 mm) on a polypropylene surface are produced in a very short graft polymerization process With aqueous acrylic acid small tufts of polyacrylate placed on the raised areas, which remain surrounded by the hydrophobic depressions. Such an area can be wetted with drops of liquids. A drop may wet one or more or many adjacent tufts; however, its spread is limited by the many narrow hydrophobic barriers between the tufts compared to a homogeneously grafted area. Such pixel arrays were used for the immobilization of peptides and proteins with densities of up to 0.25 mm. The unevenness on the polypropylene surface is not constructed but randomly distributed randomly. So here it is a random pattern of hydrophilic anchor points to which the discontinuous drops adhere. The chemical functions (carboxylic acid functions) of the polyacrylate tufts of the wetted anchors are then used for the immobilization of the dissolved probe molecules (here peptides). The application of such pixel arrays is analogous to conventional arrays.

For the preparation of structured functionalized plastic surfaces with predetermined formatting for the automated parallel in situ synthesis or immobilization of a plurality of probe molecules and their subsequent application for the simultaneous affinity enrichment and isolation of biological analyte molecules from complex mixtures, the following very simple and very efficient method is proposed ,

Hydrophobic / hydrophilic structuring of plastic surfaces A suitable plastic surface for such an array may be made of different polymers, eg of polypropylene (PP), Polyethylene (PE), polymethylpentene (PMP), polytetrafluoroethylene (PTFE, Teflon®), polyamide (PA) in different layer thicknesses and also inks, also mixed with additives, which change the properties of the base material and thus expand the application possibilities of such materials (eg with a Graphite addition provided polypropylene, which can be used because of its conductivity as a carrier material for MALDI mass spectrometry, see P103 05 957.1); PP is preferably used as the film. Layer thickness: 0.1-1.0 mm, preferably 0.15-0.3 mm

• für 60 min - 7h mit insgesamt 3x Dimethylformamid (DMF), bevorzugt 3x 30 min
• für 9 min - 1h mit 3x Ethanol, bevorzugt 3x 5 min und dann getrocknet.

The plastic surface / foil is washed before applying the mask:

• for 60 min - 7h with a total of 3x dimethylformamide (DMF), preferably 3x 30 min
• for 9 min - 1 h with 3x ethanol, preferably 3x 5 min and then dried.

On the plastic surface, a mask of an ink or a varnish is applied, leaving only the points to be modified (later the hydrophilic areas / patches) free. The ink can be, for example, an ink for permanent markers, which can be applied to the plastic surface with a plotter, printer or stamp. The pattern corresponds to the later desired appearance of the array. It can be used for the preparation of the mask but also a photoresist, both a positive and a negative photoresist. This is applied flat on the plastic surface and is exposed after drying according to the manufacturer's instructions still with UV light and then developed. In both methods, the result is a plastic surface / film that is masked at the points where the hydrophobic barriers should later be located and at the locations where the hydrophilic areas / patches are to be located later, is free of masking. The size of the areas / patches can be varied arbitrarily. Similarly, the shape of the surface (eg circle, square, rectangle or stripes). For a later manual elution of target biomolecules, a patch size between 0.3 x 0.3 mm and 2 x 2 mm is recommended. However, also much smaller, but also larger patches can be produced. According to the use of photolithography to form the mask, the smallest possible patch size is given by the wavelength used for the exposure of the photoresist. For the transfer corresponding to small volumes of liquid on and of such small patches still the appropriate tools would have to be developed. With regard to the use of such patch arrays, it is important that the liquid drops on the wetted hydrophilic patches do not come into contact with each other. For a sufficient distance (hydrophobic webs) is necessary. These should be 1/10 to 10/10 of the smallest extent of the hydrophilic patch, preferably 3 to 5/10. The size of the total surface, which can be structured, can be varied within a wide range, from a few mm ² to m ² . The preparation of correspondingly large suitable vessels for the treatment of the surfaces is not a problem.

• Ozongehalt: 1 - 10 g/h, bevorzugt 3 - 5 g/h
• Begasungszeit: 30 min - bis 10h, bevorzugt 2 - 4 h
• Ozonquelle: Labor-Ozonisator 300.5 (Sander)

The thus prepared plastic surfaces / foils are gassed with ozone.

• Ozone content: 1-10 g / h, preferably 3-5 g / h
• Fumigation time: 30 min - to 10 h, preferably 2 - 4 h
• Ozone source: laboratory ozonizer 300.5 (Sander)

The plastic surface / film was circulated for the specified fumigation at about 20 ° C with a source of the source gas mixture of ozone in oxygen. The gas mixture had the specified ozone content.

The removal of the mask from the plastic surface by a suitable solvent, e.g. with ethanol (for ink) or acetone (for photo resists and ink) and drying the plastic surface / film in the air stream.

With ozone as the gaseous reagent, arbitrarily complicated structures can be treated and hydrophilized on surfaces in a very reproducible and very uniform manner. There are no wetting problems as experienced with liquid reagents. The so treated with ozone plastic surfaces / foils now show even at the not covered during the ozonolysis sites a higher hydrophilicity, as at the covered areas. This can be achieved by wetting with e.g. Visualize water.

In comparison to the previously used methods for the production of hydrophobic / hydrophilic structures on surfaces, such as in " Manual Manufacturing of Oligonucleotides, DNA, and Protein Microchips "- Guschin, D. et al., (1997), Analytical Biochemistry, 250, 203-211 In the method described here, only a quick and easy step to be performed, the printing / stamping / plotting of the mask onto the plastic surface, has to be carried out. In the above-mentioned methods, a hydrophobic layer must first be introduced onto the glass surface in order to be able to delineate the hydrophilic structures as a result. The method described here utilizes the already hydrophobic plastic surface to form the barriers.

The activation of plastic surfaces by means of reactive ion or electron beams or plasma through a physical mask is in WO 94/05896 and also in WO 99/58245 described. However, these methods require expensive equipment, especially when larger areas are to be processed. A chemical oxidation of polypropylene by means of chromium trioxide solution (highly toxic and carcinogenic) has also been mentioned here, but no practical solution for a structured modification has been proposed.

In addition, significantly higher loads can be generated on the plastic surface during the treatment by means of ozone. For example, after an ozone treatment for 8 hours and subsequent reduction to hydroxyl groups, a loading of 10 to 13 μmol / cm ² can be achieved. By contrast, when the same material is irradiated for 8 hours in a gamma source and subsequently reduced to hydroxyl groups, only a loading of 0.1 to 0.2 nmol / cm ^{2 is obtained} . However, since the gamma radiation penetrates the mask material, no structuring is possible. Especially for the use of the affinity enrichment, however, the loading with functions and moreover with probe molecules is extremely important. The high area-specific yield of hydroxy functions in the ozone treatment is the result of the diffusion of the reactive ozone molecules into the plastic material. Depending on the treatment duration and the tightness of the plastic material, the ozone molecules penetrate into the material and therefore a correspondingly thick layer of the surface is attacked and oxidatively reacted. Herein, the surfaces described herein are different from those with O ₂ plasma-treated hydrophilized surfaces, where the reactive ones Plasma ions almost can not penetrate into the material and thus the oxidation process is limited only to the surface, which is associated with a correspondingly lower yield of hydroxyl functions.

For use in the context of the invention, the surface areas / patches which have been oxidatively hydrophilized by ozonolysis are further modified.

A) Direct functionalization of the plastic surface:

• Reduktionsmittel: 0,5 - 3 mol/l Boran-Dimethylsulfid-Komplex, bevorzugt 1 mol/l
• Lösungsmittel: Dichlormethan (DCM), Ethanol, Dimethylformamid (DMF), Methyl-tertiär-Buthylether (MTBE), bevorzugt Methyl-tertiär-Buthylether (MTBE)
• Reaktionszeit: 2 - 28h, bevorzugt 14 - 16h
• Reaktionstemperatur: 15 - 30°C, bevorzugt 20°C

Reduction of peroxides / hydroperoxides / ozonides formed during ozone treatment:

• Reducing agent: 0.5-3 mol / l borane-dimethyl sulfide complex, preferably 1 mol / l
• Solvent: dichloromethane (DCM), ethanol, dimethylformamide (DMF), methyl tertiary butyl ether (MTBE), preferably methyl tertiary butyl ether (MTBE)
• Reaction time: 2 - 28h, preferably 14 - 16h
• Reaction temperature: 15-30 ° C, preferably 20 ° C

The plastic surfaces to be reduced are covered with a solution of the specified reducing agent with the specified solvent and shaken for the specified time. After decanting the reaction solution from the plastic surface is washed with methyl tertiary-butyl ether (MTBE) and with a solution of 3mol / l NaOH / H ₂ O / 30% H ₂ O ₂ (1: 1: 1, v / v / v ) for 1 - 2 h. It is then washed with 1N NaOH, 1N HCl, water, dimethylformamide (DMF) and dichloromethane (DCM). Under these conditions, loadings of hydroxyl groups: from 0.1 to 13.5 nmol / cm ² can be achieved.

A high surface concentration of functional groups leads to an increased hydrophilicity of the surface. A high hydrophilicity leads to a stronger wetting with a hydrophilic solvent such. As water or DMF and thus to a large spread even of small volumes. For the plastic surface / film used, this means that e.g. Drops of a reaction solution with a synthetic building block very far, and with other drops, for example. mix other synthesis building blocks, if the loading of functional groups is too high. However, this is prevented by the provision of hydrophobic barriers between the individual hydrophilic patches. The reaction solution spreads only in the area of the hydrophilic patch and is stopped by it at the boundary to the hydrophobic barrier. The drops can not run into each other anymore.

These hydroxyl functions can be used to introduce other functional groups by derivatization. The procedure is analogous to the methods described in the graft polymerization for the production of e.g. primary amino functions or aldehyde groups (see below).

B) Structured graft polymerization of Kunstoffstoffoberfläche / -folie by a common polymerization process

Through the synthesis / immobilization on three-dimensional patches, a higher loading can be achieved than in the conventional immobilization of probe molecules on eg planar glasslides is possible. Up to 100 times the capacity for immobilization can be achieved with such a three-dimensional polyacrylamide gel patch, see: " Manual Manufacturing of Oligonucleotides, DNA, and Protein Microchips "- Guschin, D. et al., (1997), Analytical Biochemistry 250, 203-211 , As a result, larger amounts of probe molecules can be synthesized or immobilized in situ on the surface, which can later be used to enrich more target biomolecules. A higher sensitivity than conventional planar arrays on glass surfaces is the result.

1. 1. durch Gamma-Strahlen initiierte Pfropfpolymerisation: Monomer: siehe "Method for producing polymeric solid phase supporting materials" - Ulbricht, M., Volkmer-Engert, R., Germeroth, L., Wenschuh, H., (Anmelder: Jerini Biotools GmbH) PCT Anmeldung WO 00/12575
   Monomerkonzentration: 1 - 30 Vol%, bevorzugt 5 - 10 Vol%
   Reaktionsgefäß: Glas
   Reaktionszeit: 8 - 96h, bevorzugt 48 - 72h
   Reaktionstemperatur: 10 - 50 °C, bevorzugt 25 - 30°C
   Lösungsmittel: Lösung aus 5mM Kupfer(II)-acetat in Wasser, Methanol, Ethanol, Eisessig, Dimethylsulfoxid (DMSO) oder Dimethylformamid (DMF), bevorzugt Dimethylsulfoxid (DMSO) Strahlenquelle: Irradiator OB29 (STS, Braunschweig, D) mit einem Cs137-Isotop und einer Dosisleistung von 204,6 Gy/h. Ein aufgeführtes Monomer bzw. ein Gemisch von angegebenen Monomeren wurde in einem angegebenen Lösungsmittel bzw. einem Gemisch von angegebenen Lösungsmitteln in der angegebenen Monomerkonzentration gelöst. Die Kunststoffoberflächen/-folien wurden in einem luftdicht verschlossenen Gefäß mit einer dieser Lösungen oder mit einem flüssigen Monomer oder einem flüssigen Gemisch aus Monomeren, welche alle gelöste Kupfer(II)-salze enthielten, überschichtet und mit einem Strom aus Argon oder Stickstoff weitgehend von Sauerstoff befreit. Dann wurde das Gefäß für die angegebene Zeit bei der angegebenen Temperatur mit von der angegebenen Quelle stammenden Gamma- Strahlung bestrahlt. Anschließend wurde die Kunststoffoberfläche/-folie mit 1N Natronlauge, 1N Salzsäure, Wasser und Ethanol gewaschen und dann getrocknet.
2. 2. durch UV-Strahlung initiierte Pfropfpolymerisation: Monomer: siehe "Method for producing polymeric solid phase supporting materials" - Ulbricht, M., Volkmer-Engert, R., Germeroth, L., Wenschuh, H., (Anmelder: Jerini Biotools GmbH) PCT Anmeldung WO 00/12575
   Monomerkonzentration: 1 - 30 Vol%, bevorzugt 5 - 10 Vol%
   Reaktionsgefäß: eingeschweißt in einen entsprechend großen Polypropylenbeutel
   Bestrahlungszeit: 10 min - 24h, bevorzugt 30 min - 4h
   Reaktionstemperatur: 10 - 50 °C, bevorzugt Raumtemperatur (ca. 20°C)
   Lösungsmittel: Lösung aus 5mM Kupfer(II)-acetat in Wasser, Methanol, Ethanol, Eisessig, Dimethylsulfoxid (DMSO) oder Dimethylformamid (DMF), bevorzugt Wasser oder DMSO Photoinitiator: siehe Fouassier, J.-P., Photoinitiation, Photopolymerisation and Photocuring, Hansers Publishers, Munich, Vienne, New York, 1995 , bevorzugt Benzophenon
   Photoinitiatorkonzentration: 0,01 - 1M, bevorzugt 0,05 - 0,3M
   Abstand der Kunststoffoberfläche/-folie zur Strahlenquelle: 10 - 90cm, bevorzugt, 25cm
   W-Strahlenquelle: Quecksilberhoch- bzw. -mitteldruck-Lampen, bevorzugt wassergekühlt, z.B. Heraeus TQ 150 und Heraeus Noblelight Vorschaltgerät.
   Ein aufgeführtes Monomer bzw. ein Gemisch von angegebenen Monomeren wurde in einem angegebenen Lösungsmittel bzw. einem Gemisch von angegebenen Lösungsmitteln in der angegebenen Monomerkonzentration gelöst. Die Kunststoffoberflächen/-folien wurden in einem luftdicht verschlossenen Gefäß mit einer dieser Lösungen oder mit einem flüssigen Monomer oder einem flüssigen Gemisch aus Monomeren, die alle einen angegebenen Photoinitiator bzw. ein Gemisch aus angegebenen Photoinitiatoren in der angegebenen Photoinitiatorenkonzentration enthielten, überschichtet und mit einem Strom aus Argon oder Stickstoff weitgehend vom Sauerstoff befreit. Dann wurde das Gefäß für die angegebene Zeit bei der angegebenen Temperatur mit der von der angegebenen Quelle stammenden UV-Strahlung bestrahlt. Anschließend wurde die Kunststoffoberfläche/-folie mit 1N Natronlauge, 1N Salzsäure, Wasser und Ethanol gewaschen und dann getrocknet.
3. 3. durch thermische Zersetzung von durch Behandlung mit Ozon gebildeten Peroxiden/Hydroperoxiden/Ozoniden initiierte Pfropfpolymerisation:
   • Monomer: siehe "Method for producing polymeric solid phase supporting materials" - Ulbricht, M., Volkmer-Engert, R., Germeroth, L., Wenschuh, H., (Anmelder: Jerini Biotools GmbH) PCT Anmeldung WO 00/12575
   • Monomerkonzentration: 1 - 30 Vol%, bevorzugt 5 - 10 Vol% Reaktionsgefäß: Glas
   • Reaktionszeit: 30 min - 72h, bevorzugt 16 - 24h Reaktionstemperatur: 60 - 110 °C, bevorzugt 75 - 100°C Lösungsmittel: Lösung aus 5mM Kupfer(II)-acetat in Wasser, Methanol, Ethanol, Eisessig, Dimethylsulfoxid (DMSO) oder Dimethylformamid (DMF), bevorzugt DMSO

The structured application of a polymer layer for such 3D patcharrays could be successfully carried out on the plastic surfaces prestructured by A by graft polymerization according to three methods.

1. 1. Gamma ray initiated graft polymerization: Monomer: see "Method for producing polymeric solid phase supporting materials" - Ulbricht, M., Volkmer-Engert, R., Germeroth, L., Wenschuh, H., (Applicant: Jerini Biotools GmbH) PCT application WO 00/12575
   Monomer concentration: 1 to 30% by volume, preferably 5 to 10% by volume
   Reaction vessel: glass
   Reaction time: 8 - 96h, preferably 48 - 72h
   Reaction temperature: 10 - 50 ° C, preferably 25 - 30 ° C
   Solvent: solution of 5 mM copper (II) acetate in water, methanol, ethanol, glacial acetic acid, dimethyl sulfoxide (DMSO) or dimethylformamide (DMF), preferably dimethyl sulfoxide (DMSO) Radiation source: Irradiator OB29 (STS, Braunschweig, D) with a Cs137 Isotope and a dose rate of 204.6 Gy / h. A listed monomer or a mixture of specified monomers was dissolved in a specified solvent or a mixture of specified solvents in the indicated monomer concentration. The plastic surfaces / films were overcoated in an airtight vessel with one of these solutions or with a liquid monomer or a liquid mixture of monomers containing all dissolved cupric salts and largely with oxygen with a stream of argon or nitrogen freed. Then, the vessel was irradiated for the specified time at the indicated temperature with gamma radiation originating from the indicated source. Subsequently, the plastic surface / film was washed with 1N sodium hydroxide solution, 1N hydrochloric acid, water and ethanol and then dried.
2. 2. UV-initiated graft polymerization: Monomer: see "Method for producing polymeric solid phase supporting materials" - Ulbricht, M., Volkmer-Engert, R., Germeroth, L., Wenschuh, H., (Applicant: Jerini Biotools GmbH) PCT application WO 00/12575
   Monomer concentration: 1 to 30% by volume, preferably 5 to 10% by volume
   Reaction vessel: sealed in a correspondingly large polypropylene bag
   Irradiation time: 10 min - 24 h, preferably 30 min - 4 h
   Reaction temperature: 10 - 50 ° C, preferably room temperature (about 20 ° C)
   Solvent: Solution of 5 mM copper (II) acetate in water, methanol, ethanol, glacial acetic acid, dimethyl sulfoxide (DMSO) or dimethylformamide (DMF), preferably water or DMSO Photoinitiator: see Fouassier, J.-P., Photoinitiation, Photopolymerization and Photocuring, Hansers Publishers, Munich, Vienne, New York, 1995 , preferably benzophenone
   Photoinitiator concentration: 0.01-1M, preferably 0.05-0.3M
   Distance of the plastic surface / foil to the radiation source: 10 - 90cm, preferably, 25cm
   W radiation source: high-pressure or medium-pressure mercury lamps, preferably water-cooled, eg Heraeus TQ 150 and Heraeus Noblelight ballast.
   A listed monomer or a mixture of specified monomers was dissolved in a specified solvent or a mixture of specified solvents in the indicated monomer concentration. The plastic surfaces / films were overcoated in an airtight vessel with one of these solutions or with a liquid monomer or a liquid mixture of monomers, all containing a specified photoinitiator or a mixture of specified photoinitiators in the specified photoinitiator concentration, and with a stream largely freed of oxygen from argon or nitrogen. Then, the vessel was irradiated for the specified time at the indicated temperature with the source of UV radiation from the source. Subsequently, the plastic surface / film was washed with 1N sodium hydroxide solution, 1N hydrochloric acid, water and ethanol and then dried.
3. 3. graft polymerization initiated by thermal decomposition of peroxides / hydroperoxides / ozonides formed by treatment with ozone:
   • Monomer: see "Method for producing polymeric solid phase supporting materials" - Ulbricht, M., Volkmer-Engert, R., Germeroth, L., Wenschuh, H., (Applicant: Jerini Biotools GmbH) PCT application WO 00/12575
   • Monomer concentration: 1 to 30% by volume, preferably 5 to 10% by volume. Reaction vessel: glass
   • Reaction time: 30 minutes - 72 hours, preferably 16 - 24 hours. Reaction temperature: 60-110 ° C., preferably 75-100 ° C. Solvent: Solution of 5 mM copper (II) acetate in water, methanol, ethanol, glacial acetic acid, dimethyl sulfoxide (DMSO) or Dimethylformamide (DMF), preferably DMSO

A listed monomer or a mixture of specified monomers was dissolved in a specified solvent or a mixture of specified solvents in the indicated monomer concentration. The plastic surfaces / films were overcoated in an airtight vessel with one of these solutions or with a liquid monomer or mixture of monomers containing all dissolved cupric salts and largely oxygenated with a stream of argon or nitrogen freed. Then the vessel was heated for the indicated time at the indicated temperature. Subsequently, the plastic surface / film was washed with 1N sodium hydroxide solution, 1N hydrochloric acid, water and ethanol and then dried.

In the case of Method 1. - Gamma radiation graft initiation initiated by Method 2. - Graft polymerization initiated by UV irradiation polymerizes to a greater extent on the uncovered areas during ozonolysis than on the covered areas. On the during the Although ozonolysis of the masked areas is also polymerized to some degree, the extent is only 10 to 20% of the loading of non-ozonolysis areas / patches. This hydrophobicity difference is sufficient so that the areas covered with a mask during ozonolysis can act as a hydrophobic barrier for, for example, reaction solutions.

This result was quite surprising since normally these two graft polymerization methods are successfully performed on untreated plastic surfaces. Even without pretreatment / activation with ozone, the plastic surface is graft-polymerized throughout. That the ozonated regions tend to initiate graft polymerization to a much greater extent than the non-pretreated ones and that the resulting hydrophobicity difference is sufficient to form hydrophobic barriers was not expected to do so.

In the case of the method 3. - graft polymerization initiated by thermal decomposition of peroxides / hydroperoxides / ozonides formed by treatment with ozone - is polymerized only on the areas not covered by a mask in the previous ozonolysis. The areas / barriers / lands covered by mask during ozonolysis are not polymerized at all. The hydrophobicity difference between the hydrophilic, polymerized areas / patches of the plastic surface and the non-polymerized areas / barriers is thus much greater than in methods 1 and 3, with the same high loading of the hydrophilic areas / patches.

It should here on the difference of the 3D patches after the graft polymerization described in the variant 3 and the cross-linked polyacrylamide patcharrays produced by Guschin et al. be pointed out. The graft polymerization was carried out here without the addition of so-called crosslinking monomers (bifunctional monomers). The attached on the plastic surface polymer chains are therefore almost exclusively linear chains and are like brush hairs side by side. All chemical functions and probe molecules immobilized thereon are readily accessible to the target biomolecules. The accessibility is much less limited by diffusion than is the case with the porous cross-linked polyacrylamide patches according to Guschin et al. the case is. Although the patches prepared according to variants 1 and 2 were also graft-polymerized without crosslinker monomers, owing to the continuous generation of free radicals in the solution, significantly stronger crosslinking must be expected ( H. Waniczek in Houben-Weyl, vol. E20, p. 646, 1987 ). However, this will be significantly lower than in the specifically cross-linked gel patches according to Guschin et al.

The functionalities of the patches achieved by graft polymerization were determined indirectly after further derivatization (see below) and are between 5 and 250 nmol / cm ² . By further reducing or increasing the ozone pretreatment, the range of 0.1 to 1000 nmol / cm ^{2 can be} extended.

Further derivatization of the modified Plastic surfaces

Depending on the monomer used, after the graft polymerization, a plastic surface / film having a functionality corresponding to the monomer used, e.g. with hydroxyl, carboxyl or amino functions. The patches produced by direct functionalization after A provide hydroxyl functions.

If, however, other functional groups than those obtainable by the modification are required for in situ synthesis or immobilization on the surface, the functional groups present are derivatized in such a way that the desired functionalization is achieved. For example, the hydroxyl groups deposited on the surface by graft polymerization may be used to introduce primary amino groups as described in "Derivatized Polymeric Materials - Frank, R., Matysiak, S. (Applicant: GBF.) P (1) 96 38 085.5," PCT / EP97 / 05123 ; US 269,002 ; EP 97 943 867.8 ; Korea 10-1999-7002279 ; Norway 99 1296 ; Australia 97.45 553 described. The functionalization to primary amines via the by-pass of grafted hydroxyl groups has the advantage over the graft polymerization with a monomer already having a primary amino group that higher loadings can be achieved. This higher loading, in turn, is more favorable for the subsequent in situ synthesis or immobilization of probe molecules, and ultimately allows more sensitive detection of target biomolecules from complex mixtures.

Derivatization of hydroxyl functions:

The plastic surface / film carrying free hydroxyl groups is reacted with N, N'-carbonyldiimidazole (CDI) and so activated.
Reaction solution: 0.2 to 0.7 mol / l of N, N'-carbonyldiimidazole, preferably 0.4 to 0.5 mol / l in a suitable solvent, such as N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), acetone or Dimethylformamide (DMF), preferably dimethylformamide (DMF)
Reaction time: 60 min - 16h, preferably 4 - 6h
Reaction temperature: 20-35 ° C, preferably 25 ° C

The plastic surface / film with the directly generated or polymerized hydroxyl functions is added to the solution indicated above and shaken for the specified time. Subsequently, the plastic surface / foil is washed with the selected solvent and finally with ethanol and dried.

The thus modified and activated plastic surface / film can now be provided with the desired functional group by introducing a molecule which contains at least two functional groups, one of which has the desired later function and the other, preferably an amino group, serves to covalently attach this molecule to the activated plastic surface. Thus, the most diverse functional groups are accessible, such as primary amino functions, aldehyde functions, carboxyl functions.

Introduction of primary amino groups:

Diamines used: 1,4-diaminocyclohexane, 1,3-diaminopropane, 1,4-phenylenediamine, 1,12-diaminododecane
Reaction solution: one of the listed diamines with one
Concentration of 0.25-1.5 mol / l, preferably 0.5-1.0 mol / l Solvent: ethanol, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), water, preferably dimethylformamide (DMF)
Reaction time: 4 - 72h, preferably 16h
Reaction temperature: 20-35 ° C, preferably 25 ° C

The CDI activated plastic surface is mixed with the indicated solution of the indicated diamines and allowed to react for the specified time with shaking. It is then washed with the chosen solvent and with ethanol.

Achieved loading of the plastic surface / film with free amino functions by methods described above: 5 - 250 nmol / cm ² .

By using different long diamines, the distance of the applied probe molecules from the plastic surface can be determined. This is important, for example, when relatively large probe molecules are to be synthesized or immobilized on the surface. At too small a distance between the probe molecule and the surface, larger probe molecules may possibly be attached to the surface only with problems or not at all, for example because the binding sites in the molecule are shielded to a certain extent due to their spatial structure. In this case, one achieves incorporation of a longer spacer molecule nevertheless a connection of the probe molecule. In addition, a greater distance to the surface is also advantageous in the binding (affinity enrichment) of large target biomolecules such as proteins or even bacteriophages.

The introduction of aldehyde groups is also possible by way of derivatization of directly generated or polymerized hydroxyl groups. The activated with CDI plastic surface / film is reacted with a molecule containing a functional group, preferably an amino group, via which it can be covalently bonded to the plastic surface / film and then by a further chemical reaction, a part of the molecule is modified / modified so that an aldehyde group is formed (masked aldehyde group). For example, amino sugars, such as D (+) - glucosamine hydrochloride or D-mannosamine hydrochloride can be covalently bound to the CDI-modified plastic surface / foil via the amino function present in the molecule, and then the sugar ring can be disrupted by cleavage with sodium periodate, that free aldehyde groups arise.

Covalent Bonding of Amino Sugar to the Plastic surface:

Amino sugar: D (+) - glucosamine hydrochloride, D-mannosamine hydrochloride, preferably D (+) - glucosamine hydrochloride reaction solution: 0.1 - 1.0 mol / l solution of an amino sugar,
preferably 0.2-0.4 mol / l
Solvent: dimethyl sulfoxide (DMSO), dimethylformamide (DMF), water, preferably water
Reaction time: 60 minutes - 24 hours, preferably 16 - 18 hours

One of the amino sugars indicated above is dissolved in one of the specified solvents in the appropriate concentration. After the pH has been adjusted to pH 8 with 1N sodium hydroxide solution, the plastic surface / foil previously modified with CDI is added to this solution and allowed to react for the given time. Subsequently, the plastic surface / foil is washed with the chosen solvent and with ethanol and dried.

Periodic cleavage of the now covalently bound sugar on the plastic surface:

Reaction solution: 15-20% by weight of acetic acid in water, preferably 17% by volume
Concentration of sodium metaperiodate in the reaction solution: 0.1-0.5 mol / l, preferably 0.3 mol / l
Reaction time: 60 sec - 15 min, preferably 8 - 10 min

The derivatized with an amino sugar plastic surface / film is added to the above reaction solution and shaken for the specified time. Subsequently, the plastic surface / foil is washed with water and with ethanol.

However, it is also possible, for example, to oxidize the hydroxyl functions directly generated or polymerized on the surface with pyridinium chlorochromate (PCC) and thus to obtain aldehyde groups, but with a significantly lower yield.

Application of the probe molecules

On the functionalized plastic surfaces, or on the prepared patches, substances are either directly synthesized (e.g., by stepwise peptide synthesis) or immobilized on the surface (e.g., by chemical attachment, via a linker) or adhesively deposited by suitable synthetic methods.

For the synthesis, probe molecules of the most diverse kind are suitable, e.g. Peptides, peptide analogues such as peptoids and peptomers, PNA, oligonucleotides, DNA, organic molecules, synthetic compounds, natural products, oligosaccharides.

For example, on a surface functionalized with primary amino groups, peptides can be synthesized by means of site-directed combinatorial chemical solid-phase synthesis, such as the SPOT synthesis method. See: "Process for the rapid synthesis of supported or free peptides or oligonucleotides, sheet produced therewith, its use and apparatus for carrying out the process" - Frank, R. and Güler, S. (Applicant: sold to Jerini Biotools GmbH, 1999) P 40 27 657.9, EP 91 914 577.1 . US 5,830,637 , Australia 96.42063 , Japan 03.513 489 , Norway 93.0613 , Canada 2 090 485 , Korea 93,700 613 , Spot Synthesis: An easy technique for the positionally addressable, parallel chemical synthesis on a membrane support Frank, R. (1992) Tetrahedron, 48, 9217-9232 , For immobilization, the same probe molecules come into question as for the synthesis. As a surface can be used, for example, a plastic surface / film with free aldehyde groups to which the probe molecule, with a corresponding Hydrazino or hydroxylamino group is covalently bonded to hydrazones or oximes. However, it is also possible to achieve immobilization of a probe molecule by working with a so-called crosslinker. These may be, for example, succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). This is first bound to the amino surface of the plastic surface / film and then, due to a second reactive grouping in its molecule, allows the attachment of a probe molecule with a suitable functional group. In the case of SMCC, this is a free SH function, as occurs, for example, in cysteine residues. An overview of the manifold methods and reagents are available GT Hermanson in "Bioconjugate Techniques; 1995 Acadamic Press Inc . "or SS Wong in "Chemistry of Protein Conjugation and Cross-Linking; 1991 CRC Press Inc ,

Example: Immobilization of a probe molecule on a plastic surface with free aldehyde functions:

A plastic surface / foil structured with aldehyde functions is reacted with a peptide that has been functionalized to be present as an N-terminal hydrazine or C-terminal hydrazide. The N-hydrazino-peptide or peptide-C-hydrazide is dissolved in a citric acid-phosphate buffer, pH 6.0, and spotted onto the patches of the plastic surface with free aldehyde functions. After 2 to 20 hours reaction time, the plastic surface is washed with water, dimethylformamide (DMF) and ethanol and dried. By forming a hydrazone, a covalent bond of the peptide to the modified plastic surface / film is obtained.

Multiple affinity enrichment

The plastic surfaces / films produced in this way are suitable for the analysis of complex biological samples on the basis of the individually separated locations, on each of which a different probe molecule or another mixture of probe molecules is applied. Such complex biological samples may e.g. Proteins from extracts, antibodies from polyclonal sera, nucleic acids (DNA, RNA) from biological samples or from in vitro experiments (PCR products, restriction fragments), phage libraries and also oligosaccharide preparations.

The array of patches with the immobilized probe molecules is incubated over the whole area with the sample, so that the target biomolecules can find their binding partners and attach themselves there specifically. Excess sample is removed by washing procedures. After this affinity enrichment of the target biomolecules from the complex mixture, the target biomolecules are bound to the probe molecules according to their preferred binding partner. Now the bound target biomolecules can be detached in a site-specific manner. The detachment is carried out by applying a dissociating the interaction between the probe molecules and the target biomolecules solution. For example, a solution of guanidine hydrochloride may disrupt the interactions between plastic surface peptides as probe molecules and phage bound thereto. The phages can be detached and eluted from the surface for further analysis. The hydrophobic barriers / bridges between the hydrophilic regions / patches with the probe molecules and the target biomolecules bound to them ensure a secure separation of the individual volumes of elution solution from each other. Thus, drops of the elution solution of the one probe molecule can not mix with the drops of the elution solution, which is located on a second probe molecule, and thus also not the target biomolecules located in this elution solution. The information which target biomolecules have bound to which probe molecule is retained. After elution of the target biomolecules, the plastic surface / foil can be reused after appropriate washing steps. If one works with very sensitive target biomolecules, which may not dry out, for example, because they otherwise denature, as is the case, for example, with bacteriophages or even cells, then the wash solutions are allowed to remain on the plastic surface / foil after affinity enrichment every patch remains covered by a drop of solvent. This is automatically the case if the supernatant is allowed to drain off easily. Alternatively, liquid drops can be dispensed manually or automatically with a pipette. Due to the hydrophobic barriers / webs between the hydrophilic areas / patches, the locations with the individual probe molecules are clearly delimited. The hydrophobic webs themselves are not wetted ( illustration 1 ).

Example phage enrichment:

The same experiment was done as in Bialek et al. (Anal Bioanal Chem (2003) 376: 1006-1013 ) has been described for the use of a peptide array on a cellulose membrane.

The three ligand peptides (Ac-SFEFPPPPTDEELRL- for the EVH1 domain, Ac-GTPPPPYTVG- for the hYAP WW domain, Ac-PPPPPPPLPAPPPQP- for the rFE65 WW domain) and the Control peptide (Ac-NRPPPAVGPQPAP-) were each synthesized on one of four adjacent patches on a PP film; the 2x2 mm patches were generated by ozonolysis and thermal graft polymerization (variant 3) and amino-functionalized (about 2 nmol per patch); the peptides were described as described in Frank & Overwin ( SPOT Synthesis: Epitope Analysis with Arrays of Synthetic Peptides Prepared on Cellulose Membrane (1996) In: Methods in Molecular Biology, Vol. 66: Epitope Mapping Protocols (GE Morris, Ed.), The Humana Press Inc., Totowa, USA 149-169 .) constructed chemically from the protected Fmoc-Aminosäurebausteinen. The patch peptide array was treated in a 2 ml Eppendorf tube with 2 ml of blocking buffer for 1 h at room temperature with gentle agitation on a shaker table.

A mixture of 9x10 ⁸ T7 phages each presenting the protein domains EVH1, hYAP-WW and rFE65-WW was obtained, each with 3x10 ¹⁰ phages presenting the indifferent proteins (mouse Il-4, Fas antigen and ezrin), prepared in 1.8 ml LB medium. This mixture was mixed with 0.2 ml 5-fold concentrated blocking buffer and gently shaken for 10 min to 1 h. Then the blocked patch peptide array was added to this phage mixture and treated at 4 ° C overnight with gentle agitation on a shaking table.

Thereafter, the phage mixture was decanted and the array 5 times for 5 min with blocking buffer, 5 times for 5 min with TBST (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) and once for 5 min with TBS (25 mM Tris-HCl pH 7.4, 150 mM NaCl). The patch peptide array was placed in a Petri dish with TBS and excess TBS laterally sucked off until only the drops remained on the patches. Successively, the drop was removed on the patches, replaced by 2 .mu.l of 1M guanidinium hydrochloride, this pipette after 1 min again and the now phage-containing solution diluted in 250 .mu.l LB medium. This procedure was repeated 3 times for each patch, so that in the end each 6 μl of phage eluate from each patch was collected into the four times 250 μl volumes of LB medium.

• Ac-SFEFPPPPTDEELRL- für die EVH1-Domäne 2.5x10⁴ Phagen, 14von14 korrekt
• Ac-GTPPPPYTVG- für die hYAP-WW-Domäne; 2,4x10³ Phagen, 19von20 korrekt
• Ac-PPPPPPPLPAPPPQP für die rFE65-WW-Domäne 7x10³ Phagen, 16von20 korrekt
• Ac-NRPPPAVGPQPAP- für das Kontrollpeptid 200

100 μl of the 250 μl were used to make serial dilutions in LB medium to 1: 100, which were then plated in petri dishes on agar with E. coli BL 5615 turf. The number of phages eluted from each peptide patch was determined by the number of plaques in E. coli turf. Of 20 randomly selected phage plaques from each peptide patch, the size of the DNA insert in the phage genome was determined by PCR and agarose gel electrophoresis, respectively; this size is characteristic of the protein domain presented and known on each page. As a result, we received:

• Ac-SFEFPPPPTDEELRL- for the EVH1 domain 2.5x10 ⁴ phages, 14 of 14 correct
• Ac-GTPPPPYTVG- for the hYAP WW domain; 2.4x10 ³ phages, 19 of 20 correct
• Ac-PPPPPPPLPAPPPQP for the rFE65 WW domain 7x10 ³ phages, 16 out of 20 correctly
• Ac-NRPPPAVGPQPAP- for control peptide 200

This result is very similar to that reported by Bialek et al. with the peptide array on cellulose. The low levels of the control peptide are even better. The cellulosic array had to be cut up for this purpose, so that the phages could be eluted individually from the individual peptide spots.

Patch arrays for miniaturized parallel cell culture Our structured modified plastic surfaces were also used to advantage, for example, to seed out cells on the patches and then treat them individually by adding different reagents.

Make a patch array with adherent fibroblast cells

A polypropylene film provided with masked ozonolysis and reduction with 2x2 mm hydroxylated patches was overlaid with a 0.1 mg / ml solution of poly-L-lysine in water (Sigma-Aldrich). After draining off the excess, the wetted patches were incubated for 1 h at room temperature in a closed vessel (eg Petri dish). It was then washed well with PBS (137 mM NaCl, 2.7 mM Na ₂ HPO ₄ , 8.1 mM KCl, 1.5 mM KH ₂ PO ₄ ) and water and dried in air. Alternatively, a corresponding film in which the hydroxy-patches are preactivated with CDI may also be used; Here, the polyL-lysine is covalently linked to the patches.

These coated films can be sterilized by UV irradiation according to the manufacturer of the irradiation equipment. 3Y1 cells (a rat embryonic fibroblast cell line) were treated as described by Nagakawa and Miyamoto (Cell Struct. Funct. 23, 1998, 283-290 ). With a resuspension of these cells, one of the poly-L-lysine-coated PP films was overcoated and the excess was drained off. The cell attached to the poly-L-lysine patches was incubated for 1 to 15 hours and counted under the microscope. to To prevent dehydration, the film with the patches in the incubator was stored over a dish with the same cell culture medium. The simple procedure of overlaying and draining put nearly equal numbers of cells on each patch.

1. A) Die Substanzen werden vorher auf den Patches synthetisiert. Hierzu wird zunächst ein spezieller Linker eingeführt, der nach der Synthese und dem Ansiedeln der Zellen orthogonal durch z.B. Licht oder pH-Veränderung gespalten werden kann und so das Syntheseprodukt in das Kulturmedium auf dem Patch entlässt. Da die Verteilung der Zellen nach dem Verfahren des Überschichtens und Abtropfens sehr schnell durchführbar ist, brauchen die Linker nicht sehr stabil sein. Solche Linker sind dem Fachmann aus der Literatur zur chemischen Festphasensynthese bekannt. Die Synthese führt man analog zu Frank & overwin durch.
2. B) Die Substanzen werden vorher auf den Patches immobilisiert. Auch hier werden spezielle Verknüpfungen gewählt, die während der Ansiedelung der Zellen stabil genug sind und erst danach zur Spaltung induziert werden können. Da die Verteilung der Zellen nach dem Verfahren des Überschichtens und Abtropfens sehr schnell durchführbar ist, brauchen die Verknüpfungen nicht sehr stabil sein. Hier können z.B. licht-, pH-, enzymatisch- oder allgemein hydrolyseempfindliche Verknüpfungen genutzt werden. Solche chemischen Verknüpfungen sind dem Fachmann z.B. aus der Literatur zu Schutzgruppenchemie oder der Konjugationschemie bekannt.
3. C) Die Substanz werden nach dem Ansiedeln der Zellen dazu pipettiert. Hierzu können konventionelle Ein- oder Mehrnadel-Pipettierroboter eingesetzt werden.

The cells thus cultured can be checked for their reaction to these substances by adding external substances. There are several possibilities for that:

1. A) The substances are previously synthesized on the patches. For this purpose, a special linker is first introduced, which can be cleaved orthogonally after the synthesis and the settling of the cells by, for example, light or pH change and thus releases the synthesis product in the culture medium on the patch. Since the distribution of the cells can be performed very quickly by the overcoating and draining method, the linkers need not be very stable. Such linkers are known to those skilled in the literature for chemical solid phase synthesis. The synthesis is carried out analogously to Frank & overwin.
2. B) The substances are previously immobilized on the patches. Again, special links are chosen, which are stable enough during the settlement of the cells and only then can be induced to cleave. Since the distribution of the cells can be performed very quickly by the method of overcoating and draining, the linkages need not be very stable. Here, for example, light, pH, enzymatic or generally hydrolysis-sensitive linkages can be used. Such chemical linkages are known to the person skilled in the art, for example, from the literature on protective group chemistry or conjugation chemistry.
3. C) The substance is pipetted after the cells have settled. For this purpose, conventional single or multi-needle pipetting robots can be used.

Claims (32)

1. Hydrophobic plastic object having a planar surface that is provided with a plurality of hydrophilic areas, which are separated from each other by hydrophobic barriers of the non-modified plastic surface, their dimension being ¹/₁₀ to ¹⁰/₁₀ of the smallest dimension of a hydrophilic area, wherein the plastic surface, for the immobilization or binding of probe molecules, has been subjected to a plasma treatment or an ozonolysis for functionalization with hydroxy groups.
2. Object according to claim 1, wherein the plurality of hydrophilic areas and the hydrophobic barriers form a grid.
3. Object according to at least one of the preceding claims, wherein the plastic is polyethylene (PE), polypropylene (PP), polymethylpentene (PMP), polytetrafluoroethylene (PTFE; Teflon) or polyamide.
4. Object according to at least one of the preceding claims, wherein the object is colored.
5. Object according to at least one of the preceding claims, wherein the plastic has a content of inorganic material, especially graphite, and/or organic material, especially an inert inorganic and/or organic material.
6. Object according to at least one of the preceding claims, wherein the object is provided as a flat material, which is optionally applied to a support.
7. Object according to claim 6, wherein the object is a film.
8. Object according to at least one of the preceding claims, wherein the size of each hydrophilic area corresponds to the volume of a liquid that can be emitted by a pipette and wets the area, or is equivalent to the size of a living cell.
9. Object according to at least one of the preceding claims, wherein the hydrophilic areas carry probe molecules, which are suitable for a simultaneous affinity enrichment and isolation of analyte molecules.
10. Object according to at least one of the preceding claims, wherein the probe molecules are immobilized on the hydrophilic areas or are bound to the hydrophilic areas.
11. Object according to at least one of the preceding claims, wherein the plastic surface is functionalized for covalent immobilization or binding of probe molecules.
12. Object according to claim 11, wherein the object has been provided with a mask for functionalization, which mask is equivalent to the hydrophobic barriers to be provided or provides said hydrophobic barriers, and the masked object, insofar as it has not been masked, is subjected to a functionalization under formation of hydrophilic areas.
13. Object according to claim 11 or 12, wherein the plastic surface has been provided with hydroxy, amino (especially NH₂-groups), carboxyl, aldehyde or keto groups as functional groups.
14. Object according to claim 13, wherein the surface has been provided with 0.5 to 14 and especially 0.1 to 13.5 nanomoles of hydroxy groups/cm².
15. Object according to at least one of the claims 12 to 14, wherein the plastic surface has been further provided with functional groups by grafting a polymer layer that is provided with functional groups onto the functionalized plastic surface.
16. Object according to claim 15, wherein the grafting has been carried out in the absence of cross-linking agents, especially bi-functional monomers.
17. Object according to at least one of the preceding claims, wherein the probe molecules have been applied to the functionalized plastic surface by synthesis, by immobilization, or by adhesion.
18. Object according to claim 17, wherein the probe molecules have been applied by a stepwise synthesis.
19. Object according to claim 17, wherein the probe molecules have been immobilized by chemical binding, especially via a linker.
20. Object according to claim 17, wherein the probe molecules have been applied adhesively as to be non-washable.
21. Object according to at least one of the claims 10 to 19, wherein peptides, peptide analogues, especially peptoides and/or peptomers, PNA, oligonucleotide, DNA, organic molecules, natural products and/or oligosaccharides have been applied as probe molecules.
22. Object according to at least one of the preceding claims, wherein the probe molecules vary from hydrophilic area to hydrophilic area, or wherein different probe molecules are provided from hydrophilic area to hydrophilic area.
23. Object according to at least one of the preceding claims, the planar surface ranging in size from 1 mm² to 1m², especially from a few mm² to some cm².
24. Method for producing a plastic object having a planar surface that is provided with a plurality of hydrophilic areas, which are separated from each other by hydrophobic barriers, and functionalized to be provided with probe molecules, wherein one starts from a hydrophobic plastic, provides the object with a mask which corresponds to the hydrophobic barriers to be provided or provides said hydrophobic barriers, and functionalizes the object, insofar as it has not been masked, by hydrophilizing.
25. Method according to claim 24, wherein one hydrophilizes by providing hydroxy, amino-, carboxyl-, aldehyde- oder keto groups.
26. Method according to claim 25, wherein the area-wise hydrophilized object is subjected to a graft polymerization which graft polymerization provides hydroxy, amino, carboxyle, aldehyde or keto groups.
27. Method for producing an object according to at least one of claims 1 to 23, wherein one starts from a hydrophobic plastic, especially a plastic according to claim 3, and provides the plastic with probe molecules by providing features according to at least one of claims 1 to 23.
28. Method for producing a plastic object according to one of claims 24 to 28, wherein the object is provided with probe molecules.
29. Use of an object according to at least one of the preceding claims for a simultaneous affinity enrichment or an affinity purification and isolation of analyte particles, especially biological analyte particles, preferably molecules, proteins, antibodies, nucleic acids, phages, oligosaccharides or cells.
30. Use according to claim 29, wherein an elution solution is applied individually to each hydrophilic area after affinity enrichment or affinity purification, and the respective analyte particle is isolated.
31. Use according to claim 29 or 30, wherein the affinity enrichment or affinity purification and/or isolation of the analyte particles is carried out automatized.
32. Use according to at least one of claims 29 to 31, wherein the object that is freed from the analyte particles is reused for an affinity enrichment or affinity purification.

Images